Identification of a subpopulation of lymphocytes in human peripheral blood cytotoxic to autologous fibroblasts

A naturally occurring subpopulation of human peripheral blood lymphocytes is cytotoxic to autologous and/or allogeneic fibroblasts. The autocytotoxic lymphocytes have a receptor for the third component of complement and for aggregated gamma globulin, do not form rosettes with sheep red blood cells, and are not removed by passage through nylon. The autocytotoxic subpopulation is not present in the thymus and tonsils of normal children or in the peripheral blood of individuals with X-linked agammaglobulinemia. Fibroblast absorption experiments demonstrate that the autocytotoxic cells are "sensitized" to antigens expressed on allogeneic fibroblasts in addition to the antigens expressed on autologous cells. Some normal individuals have a second subpopulation of lymphocytes that may "regulate" the autocytotoxic cells. The relevance of these observations to the murine autocytotoxic cells is discussed.     

Active rosette forming cells as a possible functional subpopulation of human peripheral T lymphocytes.

Spontaneous E rosette forming cells (RFC) and active E rosette forming cells (ARFC) were separated individually by the rosette sedimentation technique. The responses of these cells to phytohemagglutinin (PHA) and concanavalin A (Con A) were studied. RFC exceeded ARFC in their responses to PHA at all concentrations of PHA and the maximum PHA response of RFC was significantly higher than that of ARFC. On the other hand, Con A appeared to stimulate RFC and ARFC equally at the level of an optimal dose. Therefore, the binding affinity for sheep red blood cells (SRBC) by T lymphocytes and their responsiveness to PHA stimulation may be inversely related. It is also suggested that the PHA-sensitive population may be different from the Con A-sensitive one, or that there may be heterogenous populations among human T lymphocytes in their mitogenic responses. Thus, it is possible that ARFC may be a functional subpopulation of human T cells. An additional finding is that spontaneous DNA synthesis of rosetting cells was significantly lower than that of non-rosetting cells.     

Human antibody-dependent cellular cytotoxicity. Isolation and identification of a subpopulation of peripheral blood lymphocytes which kill antibody-coated autologous target cells.

Antibody-dependent cellular cytotoxicity (ADCC), has been shown to be independent in vitro of thymus-derived lymphocytes, but the precise nature of the effector lymphocyte has not been fully clarified. To further study the identity of the ADCC effector cell type(s), peripheral blood leukocytes were purified by Ficoll-Hypaque density centrifugation and fractionated into surface immunoglobulin-positive [Ig(+)] and surface immunoglobulin-negative [Ig(-)] populations by chromatographic separation on Sephadex G-200 anti-human immunoglobulin columns. After column fractionations, the ADCC effector activity against antibody-coated autologous lymphocytes was predominantly and consistently found in the Ig(-) fraction. This latter population was then further fractionated, by rosetting techniques, into two subpopulations, The first was depleted by lymphocytes with surface receptors for sheep red blood cells [E(+)]and the second was depleted of lymphocytes with receptors for sheep red blood cell-antibody-complement [EAC-(+)]. Analysis of these populations showed that ADCC effector activity was predominantly a property of the Ig(-) lmyphocytes which are E(-) but EAC(+). These lymphocytes have been referred to as "null lymphocytes" and probably represent a subset of bone marrow-derived (B) cells. In addition, variable and low levels of ADCC activity were observed in some Ig(+) populations (B cells). Further purification of the null cell population by filtration over nylon wool columns to reduce the number of contaminating latex ingesting monocytes did not reduce ADCC effector activity. Isolated null cell ADCC effector activity was inhibited by either rabbit anti-human F(ab)2 or normal pooled rabbit gamma globulin, but not by rabbit F(ab)2 anti-human F)ab)2 or media. This supports the contention previously suggested in studies using unfractionated lymphocyte populations that the ADCC effector cell recognizes the Fc portion of the antibody molecule. The variable and low level of activity noted in the Ig(+) populations is unexplained but possibly due to a variable population of null cell-derived Ig(+) lymphocytes within the whole Ig(+) population. In conclusion, these experiments demonstrate that, in vitro, the major ADCC effector activity of circulating human peripheral blood lymphocytes resides in the Ig(-), E(-), EAC-(+) subpopulation termed "null cells." Since it has been noted that in certain disease states, such as immunodeficiency syndromes, autoimmune disorders, and neoplasms, the percentage of this population of lymphocytes in the peripheral blood is elevated, it is speculated that these cells, perhaps through their ADCC function, may play an important pathophysiologic role in these diseases.     

Cell membrane abnormality detected in erythrocytes from patients with multiple sclerosis by partition in two-polymer aqueous-phase systems

Erythrocytes from multiple sclerosis patients differ significantly (p less than 0.005) from those from controls with regard to hydrophobic affinity partition in two-polymer aqueous-phase systems containing dextran, poly(ethylene glycol) and poly (ethylene glycol)-fatty acid esters. The most likely source of the abnormality is the cell membrane.     

Normal human intestinal B lymphocytes. Increased activation compared with peripheral blood.

The state of activation of normal human intestinal mononuclear cells obtained from transplant donors was studied. Compared with PBMC, freshly isolated intestinal mononuclear cells expressed significantly more cell surface activation antigens on both B and T lymphocytes. Intestinal mononuclear cells contained significant numbers of immunoglobulin secreting cells immediately after cell separation. This population included CD5-positive B cells that secreted predominantly IgA. Cells from the large bowel consistently revealed higher numbers of IgA secreting cells than cells from the small bowel. Thus, intestinal B cells are markedly activated in vivo compared with PBMC and this increased activation correlates with increased spontaneous antibody secretion. B cells from the large intestine are more highly activated and secrete more antibody than do cells from the small intestine. The intestinal lamina propria lymphoid compartment exhibits a heightened state of activation that may be important for its distinct role in mucosal defense.     

Expansion of human cytomegalovirus-specific T lymphocytes from unfractionated peripheral blood mononuclear cells with artificial antigen-presenting cells

BACKGROUND: The aim of this study was to find a simple and feasible method for ex vivo expansion of human cytomegalovirus (CMV)-specific cytotoxic T cells from unfractionated peripheral blood mononuclear cells (PBMNCs). STUDY DESIGN AND METHODS: Unfractionated PBMNCs from three HLA-A*0201-CMV-seropositive donors were stimulated with CMVpp65(495-503) peptide-loaded HLA-A*0201-immunoglobulin fusion protein (HLA-A2-Ig) based artificial antigen-presenting cells (aAPCs) on Day 1. Once a week the CMV-specific T cells were harvested and restimulated with fresh aAPCs. T-cell cultures were maintained for 28 days and then analyzed. RESULTS: With aAPCs and starting with 1x10(7) freshly isolated PBMNCs that were less than 0.1 percent CMV-specific, more than 1x10(7) T cells with a CMV-specific frequency greater than 93 percent in all donors tested were generated. Expanded CD8+ cytotoxic T lymphocytes were functionally active and showed antigen-specific secretion of interferon-gamma and cytotoxic activity. No alloreactivity against unpulsed HLA-A*0201-positive cells was detected. CONCLUSION: Herein is reported the successful in vitro expansion of CMV-specific cytotoxic CD8+ T cells from unfractionated PBMNCs of healthy CMV-seropositive blood donors by the use of HLA-A2-Ig-based aAPCs. This study demonstrates that more than 1x10(7) CMV-specific T cells can be generated from approximately 1x10(7) unfractionated PBMNCs within 1 month under highly reproducible conditions.     

Differential processing of proenkephalin-A by human peripheral blood monocytes and T lymphocytes.

Human peripheral blood mononuclear cells are analyzed for preproenkephalin gene expression and peptide processing. Met-enkephalin immunoreactivity as detected with a specific antiserum is found in the cytoplasm of monocytes but not in T lymphocytes. Secretion of met-enkephalin was analyzed with an RIA that is specific for the met-enkephalin pentapeptide. Unfractionated PBMC spontaneously released 40 pg/ml met-enkephalin and this increased two- to fourfold after stimulation with PHA. Lower levels (less than 100 pg/ml) of met-enkephalin were detected in supernatants from purified T cells that were activated with PHA and IL-2. In contrast, stimulation of purified monocytes with LPS or PMA resulted in the release of up to 600 pg/ml of the processed peptide. To examine whether T cells can produce met-enkephalin precursor peptides, T cell conditioned media were treated with trypsin and carboxypeptidase-B, which is known to release met-enkephalin from the propeptide. This increased levels of met-enkephalin to 400 pg/ml, indicating that lymphocytes secrete the propeptide but do not process it to met-enkephalin. The 1.4-kb preproenkephalin mRNA is detected in activated blood mononuclear cells and in purified monocytes and T cells. To determine whether monocytes or lymphocytes express met-enkephalin in vivo, lymphoid tissues were analyzed by immunohistochemistry. In human spleen tissue, positive cells were found in the red pulp but not in the follicles, which is also consistent with met-enkephalin expression in monocytes. In summary, these results show that human peripheral blood mononuclear cells express preproenkephalin mRNA and that monocytes, but not T cells, process the propeptide to metenkephalin.     

颅内肿瘤淋巴细胞核仁区酸性非组蛋白的表达

目的探讨颅内肿瘤患者外周血T淋巴细胞银染核仁区酸性非组蛋白（Ag-NORs）表达特点。方法应用KL-2图像分析系统及其配套细胞培养银染等试剂和方法,分析47例颅内良性和恶性肿瘤患者外周血淋巴细胞Ag-NORs的表达,并与40名健康人和32例其他系统恶性肿瘤相比较。结果颅内良性肿瘤组患者Ag-NORs表达降低,平均值为（6.42±0.16）%,与健康人相比差异有非常显著意义;颅内恶性肿瘤组患者降低更明显［平均值(5.73±0.22)%］,与良性组相比差异有显著意义;其他系统恶性肿瘤组患者平均值［(3.68±0.19)%］比颅内恶性肿瘤组降低更加明显,差异有非常显著意义。结论外周血T淋巴细胞Ag-NORs可以作为标记颅内肿瘤的参考指标。颅内肿瘤患者的细胞免疫缺陷程度与其他系统肿瘤有差异。     

Measles infection of human mononuclear cells. I. Acute infection of peripheral blood lymphocytes and monocytes

A study of the susceptibility of human peripheral blood mononuclear cells to measles virus infection and replication is reported. Resting lymphocytes obtained from adults showed very low levels of infection and virus replication while lymphocytes activated by plant mitogens or allogenic lymphocytes supported mononuclear cells obtained from the umbilical cord of healthy neonates were more susceptible to measles virus infection than those of adults; however, activated cord lymphocytes supported viral replication in the range observed with adult activated lymphocytes. Monocytes obtained from adults were relatively resistant to measles virus infection and replication while neonatal cord blood monocytes supported viral replication to the degree observed with activated lymphocytes. It is hypothesized that infection of acitivated lymphocytes may explain the depression of cell-mediated immunity seen during acute measles virus infection. The significance of the finding that neonatal monocytes are more susceptible to viral infection and replication than adult monocytes is discussed.     

Production of predominantly polymeric IgA by human peripheral blood lymphocytes stimulated in vitro with mitogens

Human peripheral blood lymphocytes (PBL) were cultured for various time periods (up to 8 d) in the presence of pokeweed mitogen (PWM), lipopolysaccharide, or Epstein-Barr virus. Cell-free supernates were fractionated on a standardized ultrogel AcA 22 column and the proportion of polymeric and monomeric IgA was determined by radioimmunoassay. The results demonstrate that PBL stimulated with these mitogens produce IgM and IgG with molecular characteristics identical to those found in serum, but that the IgA produced is predominantly of the polymeric type. To prove that this IgA represented disulfide bond-linked polymers rather than aggregated monomers, we have demonstrated that the high molecular weight IgA (a) maintains its polymeric form upon treatment with acidic buffers, (b) contains J chain, a glycoprotein associated only with polymeric immunoglobulins, and (c) dissociates to the monomeric form upon reduction of disulfide bonds. After 1 wk in culture, approximately 60% of the PWM-stimulated cells that contained IgA were positive for IgA2, whereas 40% were IgA1 positive as determined by immunofluorescence. Therefore, peripheral blood contains a population of lymphocytes with the potential to display, after appropriate stimulation and differentiation, characteristics similar to IgA cells found in external secretory tissues. The demonstration of the presence of such cells in the peripheral circulation suggests that these cells are precursors of IgA- producing plasma cells with the potential to populate mucosal tissues.     

Retinoid protection against x-ray-induced chromatid damage in human peripheral blood lymphocytes.

Oral administration of isotretinoin (13-cis retinoic acid) was shown previously (Kraemer, K. H., J. J. DiGiovanna, A. N. Moshell, R. E. Tarone, and G. L. Peck. 1988. N. Engl. J. Med. 318:1633-1637) to reduce the frequency of skin cancers in xeroderma pigmentosum (XP) patients. The mechanism of protection was unclear. In the present study, x-ray-induced chromatid damage in PHA-stimulated blood lymphocytes from five XP patients receiving isotretinoin was approximately half that in blood samples from the same patients before or subsequent to treatment. The x-ray-induced chromatid damage in blood lymphocytes from a normal control was reduced significantly by cocultivation with blood or plasma from an XP patient receiving isotretinoin or by addition of 10(-6) M isotretinoin to cultures 1 h before x-irradiation. A similar reduction in x-ray-induced chromatid damage was reported previously by adding to the culture medium, mannitol, a scavenger of the free hydroxyl radical, or catalase, which decomposes hydrogen peroxide; both of these products are generated during ionizing radiation. The present observations suggest that isotretinoin acts as a scavenger of such radiation products, thereby providing protection against x-ray-induced chromatid damage.     

Phenotypic analysis of human peripheral blood lymphocytes by automatic sampling flow cytometry after stimulation with mitogens or allogeneic cells

Summary Human peripheral blood lymphocyte (PBL) phenotypes have been analyzed before and after stimulation with phytohemagglutinin (PHA), concanavalin A (ConA) and pokeweed mitogen (PWM) for 3 days and in mixed lymphocyte culture (MLC) for 7 days. PBL labeled with each of 10 fluorescent monoclonal antibodies were automatically sampled for flow cytometry from 96-well microtiter plates using a microsample delivery system. The reference phenotypic ranges were determined in fresh cells and control cultures. PHA was mostly mitogenic for T PBL bearing the CD3, CD5, CD7, CD8 and CD25 differentiation clusters, and a low density of CD1 and CD4 had a small effect on human natural killer cells (HNK) and also did not stimulate B (CD19) and HLA-DR⁺ PBL. There was an incomplete phenotypic overlapping between PHA- and ConA-stimulated cultures, ConA being more mitogenic for CD4 and less mitogenic for CD8 PBL. The mitogenic effect of PWM was evident on CD3, CD5, CD7, CD4, CD25 and CD8, but not on HNK, HLA-DR and CD19 B PBL, which presumably had already differentiated into antibody-secreting cells. After MLC stimulation all T, B and HNK PBL subsets tested were increased, but the cells bearing CD1, CD4, CD5, CD7, CD25, HNK, CD19 and HLA-DR had the greatest proliferation with respect to the unmixed control PBL. The present approach to the phenotyping of PBL subsets could offer more complete and accurate data for monitoring and follow-up of patients in transplantation and immunopathology hospital wards. This work was supported in part by grants from theConsiglio Nazionale delle Ricerche (CNR), Roma, Italy,Progetto Finalizzato ‘Oncologia’, theAssociazione Italiana per la Ricerca sul Cancro (AIRC), and theAssociazione per le Ricerche Biomediche, Verona, Italy.     

Specific HIV-1 env gene silencing by small interfering RNAs in human peripheral blood mononuclear cells

RNA interference (RNAi) is triggered by the presence of a double-stranded RNA (dsRNA) in the cell, and results in the silencing of homologous gene expression by the specific degradation of an mRNA containing the same sequence. dsRNA-mediated RNAi can be used in a wide variety of eucaryotes to induce the sequence-specific inhibition of gene expression. Synthetic 21-23 nucleotide (nt) small interfering RNAs (siRNAs) with 2-nt 3' overhangs were recently found to mediate efficient sequence-specific mRNA degradation in mammalian cells. Here, we show that synthetic siRNAs targeted against the viral structural Env proteins encoded by HIV-1 can specifically suppress the expression of HIV-1 genes. The siRNA-mediated RNAi also had advantages over antisense RNA-mediated inhibition, in terms of both the ease of designing effective antiviral agents and their potency. Especially, our best env-specific siRNAs, E7145 targeted to the central region of the V3 loop and E7490 targeted to the CD4 binding site of conserved regions on gp120, significantly inhibited the HIV-1 gene expression. Furthermore, E7145 and E7490 were effective against HIV-1(NL4-3) replication in PBMCs for a relatively long time (14 days). Therefore, the use of synthetic siRNAs provides a simple, rapid, and cost-effective tool for new anti-HIV-1 gene therapeutics.     

Cocaine-mediated suppression of superoxide production by human peripheral blood mononuclear cells.

Cocaine, like opiates, modulates a variety of immune functions. In the present study, we investigated the effect of cocaine on superoxide anion (O2-) production, an index of a microbicidal activity, by cultured human peripheral blood mononuclear cells. Release of O2- was measured by superoxide dismutase-inhibitable reduction of ferricytochrome C in response to phorbol myristate acetate. Peripheral blood mononuclear cells cultured in the presence of cocaine (1 microM) for 48 hr released less (P less than .05) O2- than did nontreated control cells (95.1 +/- 10.2 vs. 57.9 +/- 6.6 nmol/10(7) cells/60 min, respectively). This suppressive effect was dose-dependent. Antibodies to transforming growth factor-beta, a cytokine inhibitory of monocyte O2- production, abrogated (P less than .01) cocaine-mediated suppression, suggesting that transforming growth factor-beta is involved in the suppression. Also, naloxone blocked (P less than .01) the suppressive effects of both cocaine and transforming growth factor-beta on O2- production, suggesting that the suppressive mechanism is naloxone-sensitive.     

Immunoglobulin-bearing and complement-receptor lymphocytes constitute the same population in human peripheral blood.

Complement-receptor lymphocytes have generally been considered to be a subpopulation of bone-marrow derived (B) lymphocytes. However, the present studies show that essentially all cells with integral surface immunoglobulin from normal human peripheral blood bear receptors for the third component of complement. Moreover, after removal of phagocytes, all cells with complement receptors bear surface Ig. Thus, circulating B cells and complement-receptor lymphocytes are the same population.